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The apolipoprotein E4 (APOE4) allele represents the major genetic risk factor for Alzheimer's disease (AD). In contrast, APOE2 is known to lower the AD risk, while APOE3 is defined as risk neutral. APOE plays a prominent role in the bioenergetic homeostasis of the brain, and early-stage metabolic changes have been detected in the brains of AD patients. Although APOE is primarily expressed by astrocytes in the brain, neurons have also been shown as source for APOE. However, the distinct roles of the three APOE isoforms in neuronal energy homeostasis remain poorly understood. In this study, we generated pure human neurons (iN cells) from APOE-isogenic induced pluripotent stem cells (iPSCs), expressing either APOE2, APOE3, APOE4, or carrying an APOE knockout (KO) to investigate APOE isoform-specific effects on neuronal energy metabolism. We showed that endogenously produced APOE4 enhanced mitochondrial ATP production in APOE-isogenic iN cells but not in the corresponding iPS cell line. This effect neither correlated with the expression levels of mitochondrial fission or fusion proteins nor with the intracellular or secreted levels of APOE, which were similar for APOE2, APOE3, and APOE4 iN cells. ATP production and basal respiration in APOE-KO iN cells strongly differed from APOE4 and more closely resembled APOE2 and APOE3 iN cells, indicating a gain-of-function mechanism of APOE4 rather than a loss-of-function. Taken together, our findings in APOE isogenic iN cells reveal an APOE genotype-dependent and neuron-specific regulation of oxidative energy metabolism.

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Tauopathies are age-associated neurodegenerative diseases whose mechanistic underpinnings remain elusive, partially due to a lack of appropriate human models. Here, we engineered human induced pluripotent stem cell (hiPSC)-derived neuronal lines to express 4R Tau and 4R Tau carrying the P301S MAPT mutation when differentiated into neurons. 4R-P301S neurons display progressive Tau inclusions upon seeding with Tau fibrils and recapitulate features of tauopathy phenotypes including shared transcriptomic signatures, autophagic body accumulation, and reduced neuronal activity. A CRISPRi screen of genes associated with Tau pathobiology identified over 500 genetic modifiers of seeding-induced Tau propagation, including retromer VPS29 and genes in the UFMylation cascade. In progressive supranuclear palsy (PSP) and Alzheimer's Disease (AD) brains, the UFMylation cascade is altered in neurofibrillary-tangle-bearing neurons. Inhibiting the UFMylation cascade in vitro and in vivo suppressed seeding-induced Tau propagation. This model provides a robust platform to identify novel therapeutic strategies for 4R tauopathy.

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Our study explores the pressing issue of micro- and nanoplastics (MNPs) inhalation and their subsequent penetration into the brain, highlighting a significant environmental health concern. We demonstrate that MNPs can indeed penetrate murine brain, warranting further investigation into their neurotoxic effects in humans. We then proceed to test the impact of MNPs at environmentally relevant concentrations, with focusing on variations in size and shape. Our findings reveal that these MNPs induce oxidative stress, cytotoxicity, and neurodegeneration in human neurons, with cortical neurons being more susceptible than nociceptors. Furthermore, we examine the role of biofilms on MNPs, demonstrating that MNPs can serve as a vehicle for pathogenic biofilms that significantly exacerbate these neurotoxic effects. This sequence of investigations reveals that minimal MNPs accumulation can cause oxidative stress and neurodegeneration in human neurons, significantly risking brain health and highlights the need to understand the neurological consequences of inhaling MNPs. Overall, our developed in vitro testing battery has significance in elucidating the effects of environmental factors and their associated pathological mechanisms in human neurons.

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Human pluripotent stem cell (hPSC) models provide unprecedented opportunities to study human neurological disorders by recapitulating human-specific disease mechanisms. In particular, hPSC-based human-animal brain chimeras enable the study of human cell pathophysiology in vivo. In chimeric brains, human neural and immune cells can maintain human-specific features, undergo maturation, and functionally integrate into host brains, allowing scientists to study how human cells impact neural circuits and animal behaviors. The emerging human-animal brain chimeras hold promise for modeling human brain cells and their interactions in health and disease, elucidating the disease mechanism from molecular and cellular to circuit and behavioral levels, and testing the efficacy of cell therapy interventions. Here, we discuss recent advances in the generation and applications of using human-animal chimeric brain models for the study of neurological disorders, including disease modeling and cell therapy.

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Besides respiratory illness, SARS-CoV-2, the causative agent of COVID-19, leads to neurological symptoms. The molecular mechanisms leading to neuropathology after SARS-CoV-2 infection are sparsely explored. SARS-CoV-2 enters human cells via different receptors, including ACE-2, TMPRSS2, and TMEM106B. In this study, we used a human-induced pluripotent stem cell-derived neuronal model, which expresses ACE-2, TMPRSS2, TMEM106B, and other possible SARS-CoV-2 receptors, to evaluate its susceptibility to SARS-CoV-2 infection. The neurons were exposed to SARS-CoV-2, followed by RT-qPCR, immunocytochemistry, and proteomic analyses of the infected neurons. Our findings showed that SARS-CoV-2 infects neurons at a lower rate than other human cells; however, the virus could not replicate or produce infectious virions in this neuronal model. Despite the aborted SARS-CoV-2 replication, the infected neuronal nuclei showed irregular morphology compared to other human cells. Since cytokine storm is a significant effect of SARS-CoV-2 infection in COVID-19 patients, in addition to the direct neuronal infection, the neurons were treated with pre-conditioned media from SARS-CoV-2-infected lung cells, and the neuroproteomic changes were investigated. The limited SARS-CoV-2 infection in the neurons and the neurons treated with the pre-conditioned media showed changes in the neuroproteomic profile, particularly affecting mitochondrial proteins and apoptotic and metabolic pathways, which may lead to the development of neurological complications. The findings from our study uncover a possible mechanism behind SARS-CoV-2-mediated neuropathology that might contribute to the lingering effects of the virus on the human brain.

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The MMP-9-1562C/T polymorphism exerts an impact on the occurrence and progression of numerous disorders affecting the central nervous system. Using luciferase assays and Q-RT-PCR technique, we have discovered a distinct allele-specific influence of the MMP-9-1562C/T polymorphism on the MMP-9 (Extracellular Matrix Metalloproteinase-9) promoter activity and the expression of MMP-9 mRNA in human neurons derived from SH-SY5Y cells. Subsequently, by employing a pull-down assay paired with mass spectrometry analysis, EMSA (Electromobility Shift Assay), and EMSA supershift techniques, as well as DsiRNA-dependent gene silencing, we have elucidated the mechanism responsible for the allele-specific impact of the MMP-9-1562C/T polymorphism on the transcriptional regulation of the MMP-9 gene. We have discovered that the activity of the MMP-9 promoter and the expression of MMP-9 mRNA in human neurons are regulated in a manner that is specific to the MMP-9-1562C/T allele, with a stronger upregulation being attributed to the C allele. Furthermore, we have demonstrated that the allele-specific action of the MMP-9-1562C/T polymorphism on the neuronal MMP-9 expression is related to HDAC1 (Histone deacetylase 1) and ZNF384 (Zinc Finger Protein 384) transcriptional regulators. We show that HDAC1 and ZNF384 bind to the C and the T alleles differently, forming different regulatory complexes in vitro. Moreover, our data demonstrate that HDAC1 and ZNF384 downregulate MMP-9 gene promoter activity and mRNA expression in human neurons acting mostly via the T allele.

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Grid cells in the entorhinal cortex demonstrate spatially periodic firing, thought to provide a spatial map on behaviorally relevant length scales. Whether such periodicity exists for behaviorally relevant time scales in the human brain remains unclear. We investigate neuronal firing during a temporally continuous experience by presenting 14 neurosurgical patients with a video while recording neuronal activity from multiple brain regions. We report on neurons that modulate their activity in a periodic manner across different time scales-from seconds to many minutes, most prevalently in the entorhinal cortex. These neurons remap their dominant periodicity to shorter time scales during a subsequent recognition memory task. When the video is presented at two different speeds, a significant percentage of these temporally periodic cells (TPCs) maintain their time scales, suggesting a degree of invariance. The TPCs' temporal periodicity might complement the spatial periodicity of grid cells and together provide scalable spatiotemporal metrics for human experience.

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Time and space are primary dimensions of human experience. Separate lines of investigation have identified neural correlates of time and space, yet little is known about how these representations converge during self-guided experience. Here, 10 subjects with intracranially implanted microelectrodes play a timed, virtual navigation game featuring object search and retrieval tasks separated by fixed delays. Time cells and place cells activate in parallel during timed navigation intervals, whereas a separate time cell sequence spans inter-task delays. The prevalence, firing rates, and behavioral coding strengths of time cells and place cells are indistinguishable-yet time cells selectively remap between search and retrieval tasks, while place cell responses remain stable. Thus, the brain can represent time and space as overlapping but dissociable dimensions. Time cells and place cells may constitute a biological basis for the cognitive map of spatiotemporal context onto which memories are written.

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Secretory pathways within dendrites of neurons have been proposed for local transport of newly synthesized proteins. However, little is known about the dynamics of the local secretory system and whether the organelles are transient or stable structures. Here, we quantify the spatial and dynamic behavior of dendritic Golgi and endosomes during differentiation of human neurons generated from induced pluripotent stem cells (iPSCs). In early neuronal development, before and during migration, the entire Golgi apparatus transiently translocates from the soma into dendrites. In mature neurons, dynamic Golgi elements, containing cis and trans cisternae, are transported from the soma along dendrites, in an actin-dependent process. Dendritic Golgi outposts are dynamic and display bidirectional movement. Similar structures were observed in cerebral organoids. Using the retention using selective hooks (RUSH) system, Golgi resident proteins are transported efficiently into Golgi outposts from the endoplasmic reticulum. This study reveals dynamic, functional Golgi structures in dendrites and a spatial map for investigating dendrite trafficking in human neurons.

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Toxoplasma gondii infects approximately one-third of the world's population resulting in a chronic infection with the parasite located in cysts in neurons in the brain. In most immunocompetent hosts the chronic infection is asymptomatic, but several studies have found correlations between Toxoplasma seropositivity and neuropsychiatric disorders, including Schizophrenia, and some other neurological disorders. Host-parasite interactions of bradyzoites in cysts in neurons is not well understood due in part to the lack of suitable in vitro human neuronal models. The advent of stem cell technologies in which human neurons can be derived in vitro from human induced pluripotent stem cells (hiPSCs) or direct conversion of somatic cells generating induced neurons (iNs), affords the opportunity to develop in vitro human neuronal culture systems to advance the understanding of T. gondii in human neurons. Human neurons derived from hiPSCs or iNs, generate pure human neuron monolayers that express differentiated neuronal characteristics. hiPSCs also generate 3D neuronal models that better recapitulate the cytoarchitecture of the human brain. In this review, an overview of iPSC-derived neurons and iN protocols leading to 2D human neuron cultures and hiPSC-derived 3D cerebral organoids will be given. The potential applications of these 2D and 3D human neuronal models to address questions about host-parasite interactions of T. gondii in neurons and the parasite in the CNS, will be discussed. These human neuronal in vitro models hold the promise to advance the understanding of T. gondii in human neurons and to improve the understanding of neuropathogenesis of chronic toxoplasmosis.

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Mutations in the MECP2 gene underlie a spectrum of neurodevelopmental disorders, most commonly Rett syndrome (RTT). We ask whether MECP2 mutations interfere with human astrocyte developmental maturation, thereby affecting their ability to support neurons. Using human-based models, we show that RTT-causing MECP2 mutations greatly impact the key role of astrocytes in regulating overall brain bioenergetics and that these metabolic aberrations are likely mediated by dysfunctional mitochondria. During post-natal maturation, astrocytes rely on neurons to induce their complex stellate morphology and transcriptional changes. While MECP2 mutations cause cell-intrinsic aberrations in the astrocyte transcriptional landscape, surprisingly, they do not affect the neuron-induced astrocyte gene expression. Notably, however, astrocytes are unable to develop complex mature morphology due to cell- and non-cell-autonomous aberrations caused by MECP2 mutations. Thus, MECP2 mutations critically impact key cellular and molecular features of human astrocytes and, hence, their ability to interact and support the structural and functional maturation of neurons.

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Latency and reactivation in neurons are critical aspects of VZV pathogenesis that have historically been difficult to investigate. Viral genomes are retained in many human ganglia after the primary infection, varicella; and about one-third of the naturally infected VZV seropositive population reactivates latent virus, which most often clinically manifests as herpes zoster (HZ or Shingles). HZ is frequently complicated by acute and chronic debilitating pain for which there remains a need for more effective treatment options. Understanding of the latent state is likely to be essential in the design of strategies to reduce reactivation. Experimentally addressing VZV latency has been difficult because of the strict human species specificity of VZV and the fact that until recently, experimental reactivation had not been achieved. We do not yet know the neuron subtypes that harbor latent genomes, whether all can potentially reactivate, what the drivers of VZV reactivation are, and how immunity interplays with the latent state to control reactivation. However, recent advances have enabled a picture of VZV latency to start to emerge. The first is the ability to detect the latent viral genome and its expression in human ganglionic tissues with extraordinary sensitivity. The second, the subject of this chapter, is the development of in vitro human neuron systems permitting the modeling of latent states that can be experimentally reactivated. This review will summarize recent advances of in vitro models of neuronal VZV latency and reactivation, the limitations of the current systems, and discuss outstanding questions and future directions regarding these processes using these and yet to be developed models. Results obtained from the in vitro models to date will also be discussed in light of the recent data gleaned from studies of VZV latency and gene expression learned from human cadaver ganglia, especially the discovery of VZV latency transcripts that seem to parallel the long-studied latency-associated transcripts of other neurotropic alphaherpesviruses.

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Temporal lobe epilepsy is the fourth most common neurological disorder, with about 40% of patients not responding to pharmacological treatment. Increased cellular loss is linked to disease severity and pathological phenotypes such as heightened seizure propensity. While the hippocampus is the target of therapeutic interventions, the impact of the disease at the cellular level remains unclear. Here, we show that hippocampal granule cells change with disease progression as measured in living, resected hippocampal tissue excised from patients with epilepsy. We show that granule cells increase excitability and shorten response latency while also enlarging in cellular volume and spine density. Single-nucleus RNA sequencing combined with simulations ascribes the changes to three conductances: BK, Cav2.2, and Kir2.1. In a network model, we show that these changes related to disease progression bring the circuit into a more excitable state, while reversing them produces a less excitable, "early-disease-like" state.

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Down syndrome (DS), driven by an extra copy of chromosome 21 (HSA21), and fragile X syndrome (FXS), driven by loss of the RNA-binding protein FMRP, are two common genetic causes of intellectual disability and autism. Based upon the number of DS-implicated transcripts bound by FMRP, we hypothesize that DS and FXS may share underlying mechanisms. Comparing DS and FXS human pluripotent stem cell (hPSC) and glutamatergic neuron models, we identify increased protein expression of select targets and overlapping transcriptional perturbations. Moreover, acute upregulation of endogenous FMRP in DS patient cells using CRISPRa is sufficient to significantly reduce expression levels of candidate proteins and reverse 40% of global transcriptional perturbations. These results pinpoint specific molecular perturbations shared between DS and FXS that can be leveraged as a strategy for target prioritization; they also provide evidence for the functional relevance of previous associations between FMRP targets and disease-implicated genes.

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Human-induced pluripotent stem cells (hiPSCs) represent one of the main and powerful tools for the in vitro modeling of neurological diseases. Standard hiPSC-based protocols make use of animal-derived feeder systems to better support the neuronal differentiation process. Despite their efficiency, such protocols may not be appropriate to dissect neuronal specific properties or to avoid interspecies contaminations, hindering their future translation into clinical and drug discovery approaches. In this work, we focused on the optimization of a reproducible protocol in feeder-free conditions able to generate functional glutamatergic neurons. This protocol is based on a generation of neuroprecursor cells differentiated into human neurons with the administration in the culture medium of specific neurotrophins in a Geltrex-coated substrate. We confirmed the efficiency of this protocol through molecular analysis (upregulation of neuronal markers and neurotransmitter receptors assessed by gene expression profiling and expression of the neuronal markers at the protein level), morphological analysis, and immunfluorescence detection of pre-synaptic and post-synaptic markers at synaptic boutons. The hiPSC-derived neurons acquired Ca2+-dependent glutamate release properties as a hallmark of neuronal maturation. In conclusion, our study describes a new methodological approach to achieve feeder-free neuronal differentiation from hiPSC and adds a new tool for functional characterization of hiPSC-derived neurons.

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Tau is a microtubule-binding protein expressed in neurons, and the equal ratios between 4-repeat (4R) and 3-repeat (3R) isoforms are maintained in normal adult brain function. Dysregulation of 3R:4R ratio causes tauopathy, and human neurons that recapitulate tau isoforms in health and disease will provide a platform for elucidating pathogenic processes involving tau pathology. We carried out extensive characterizations of tau isoforms expressed in human neurons derived by microRNA-induced neuronal reprogramming of adult fibroblasts. Transcript and protein analyses showed that miR neurons expressed all six isoforms with the 3R:4R isoform ratio equivalent to that detected in human adult brains. Also, miR neurons derived from familial tauopathy patients with a 3R:4R ratio altering mutation showed increased 4R tau and the formation of insoluble tau with seeding activity. Our results collectively demonstrate the utility of miRNA-induced neuronal reprogramming to recapitulate endogenous tau regulation comparable with the adult brain in health and disease.

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A major obstacle in studying human central nervous system (CNS) diseases is inaccessibility to the affected tissue and cells. Even in limited cases where tissue is available through surgical interventions, differentiated neurons cannot be maintained for extended time frames, which is prohibitive for experimental repetition and scalability. Advances in methodologies for reprogramming human somatic cells into induced pluripotent stem cells (iPSC) and directed differentiation of human neurons in culture now allow access to physiological and disease relevant cell types. In particular, patient iPSC-derived neurons represent unique ex vivo neuronal networks that allow investigating disease genetic and molecular pathways in physiologically accurate cellular microenvironments, importantly recapitulating molecular and cellular phenotypic aspects of disease. Generation of functional neural cells from iPSCs relies on manipulation of culture formats in the presence of specific factors that promote the conversion of pluripotent stem cells into neurons. To this end, several experimental protocols have been developed. Direct differentiation of stem cells into post-mitotic neurons is usually associated with low throughput, low yield, and high technical variability. Instead, methods relying on expansion of the intermediate neural progenitor cells (NPCs) show incredible potential for posterior generation of suitable neuronal cultures for cellular and biochemical assays, as well as drug screening. NPCs are expandable, self-renewable multipotent cells that can differentiate into astrocytes, oligodendrocytes, and electrically active neurons. Here, we describe a protocol for generating iPSC-derived NPCs via formation of neural aggregates and selection of NPC precursor neural rosettes, followed by a simple and reproducible method for generating a mixed population of cortical-like neurons through growth factor withdrawal. Implementation of this protocol has the potential to advance the goals of precision medicine research for both neurological and psychiatric disorders.

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Heterozygous loss-of-function (LoF) mutations in SETD1A, which encodes a subunit of histone H3 lysine 4 methyltransferase, cause a neurodevelopmental syndrome and increase the risk for schizophrenia. Using CRISPR-Cas9, we generate excitatory/inhibitory neuronal networks from human induced pluripotent stem cells with a SETD1A heterozygous LoF mutation (SETD1A+/-). Our data show that SETD1A haploinsufficiency results in morphologically increased dendritic complexity and functionally increased bursting activity. This network phenotype is primarily driven by SETD1A haploinsufficiency in glutamatergic neurons. In accordance with the functional changes, transcriptomic profiling reveals perturbations in gene sets associated with glutamatergic synaptic function. At the molecular level, we identify specific changes in the cyclic AMP (cAMP)/Protein Kinase A pathway pointing toward a hyperactive cAMP pathway in SETD1A+/- neurons. Finally, by pharmacologically targeting the cAMP pathway, we are able to rescue the network deficits in SETD1A+/- cultures. Our results demonstrate a link between SETD1A and the cAMP-dependent pathway in human neurons.

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Alzheimer's disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aß) in the brain, which leads to progressive cognitive deficits and dementia. Aß is generated by the successive cleavage of the amyloid precursor protein (APP), first by ß-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aß aggregation and the development of AD. In vitro studies have demonstrated that Aß assemblies spark a feed-forward loop heightening Aß production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aß enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aß42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aß or to activate Go protein, we have found that treatment with aggregated Aß fails to increase colocalization of APP with BACE1 indicating that Aß-APP/Go signaling is involved in this process. Moreover, inhibition of Gßγ subunit signaling with ßARKct or gallein prevents Aß-dependent interaction of APP and BACE1 in endosomes, ß-processing of APP, and intracellular accumulation of Aß42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aß pathology in the early stages of AD and suggest that gallein could have therapeutic potential.

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Iron deposition in the brain begins early in multiple sclerosis (MS) and continues unabated. Ferrous iron is toxic to neurons, yet the therapies used in MS do not counter iron neurotoxicity. Extracts of Hibiscus sabdariffa (HS) are used in many cultures for medicinal purposes. We collected a distinct HS extract and found that it abolished the killing of neurons by iron in culture; medications used in MS were ineffective when similarly tested. Neuroprotection by HS was not due to iron chelation or anthocyanin content. In free radical scavenging assays, HS was equipotent to alpha lipoic acid, an anti-oxidant being tested in MS. However, alpha lipoic acid was only modestly protective against iron-mediated killing. Moreover, a subfraction of HS without radical scavenging activity negated iron toxicity, whereas a commercial hibiscus preparation with anti-oxidant activity could not. The idea that HS might have altered properties within neurons to confer neuroprotection is supported by its amelioration of toxicity caused by other toxins: beta-amyloid, rotenone and staurosporine. Finally, in a mouse model of MS, HS reduced disability scores and ameliorated the loss of axons in the spinal cord. HS holds therapeutic potential to counter iron neurotoxicity, an unmet need that drives the progression of disability in MS.

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